1. Field of the Invention
The present invention relates generally to immunological assay techniques for determining the presence of an analyte in serum. More particularly, this invention relates to processes that are useful in determining very small elevations of CK-MB in patients who have experienced acute myocardial infarction.
2. Description of the Prior Art
Creatine Kinase (hereinafter CK) occurs in animal body fluids and tissues in several forms including isoenzymes CK-MM, CK-BB, CK-MB, CK-BB.IgG, Mitochondrial CK and other short lived variants of CK-MM and CK-MB. While the exact structures of some of these isoenzymes have not been identified, it is clear that each of them includes two of the subunits of CK, the B subunit and the M subunit. The level of circulating CK-MB has proved to be the most reliable indicator myocardial damage, and the level of CK-MB in circulating blood increases when the necrosis of mycardial cells rich in CK-MB occurs.
Several methods, including both immunological and non-immunological methods, have been described for measuring CK-MB levels. Many of these methods such as electrophoresis, column chromatography, immunoinhibition, and B-unit radioimmunoassay (hereinafter RIA) have not been satisfactory in providing the accurate and rapid measurement of CK-MB which is essential for immediate treatment of patients suffering from life threatening episodes of Acute Myocardial Infarction (hereinafter AMI). It is estimated that an accurate detection of one to two nanograms (ng) per milliliter (ml) increase in the level of CK-MB in the patients blood is required to make the earliest possible diagnosis of AMI. Typically, when the analytical sensitivity of a CK-MB test method is increased in an attempt to measure such small elevation, the specifically is decreased due to the interference from isoenzymes of CK other than CK-MB and other non-specific effects. The inaccuracy in measurement of the small elevations of circulating CK-MB, so essential for accurate diagnosis of AMI is caused by the limitations of specifically and sensitivity.
The difficulties encountered in accurately measuring small elevations CK-MB by non-immunological methods arise due to poor separation and recovery of the CK-MB isoenzymes in specimens that contain several forms of CK. The non-immunological methods are based on the measurements of enzymatic activity of CK and require the separation of the CK-MB from other isoenzymes. These methods include electrophoresis or column chromatography and are described in U.S. Pat. Nos. 4,105,499; and 4,046,634. They produce inaccurate results due to low sensitivity, incomplete separation, poor analytical recovery, and artifacts. The selective activation procedure, described in U.S. Pat. No. 3,994,783, was a non-immunological procedure not requiring separation. However, it was quickly determined that the method did not have the sensitivity and accuracy required for clinical use.
Immunological methods are difficult to use because antibodies that are raised specifically against CK-MB cross react with other isoenzymes of CK due to the presence of either a B subunit or an M subunit in those isoenzymes. Yet, several immunological methods have been introduced as an attempt to increase sensitivity and specificity of the CK-MB measurement by utilizing antibodies raised against either CK-MM or CK-BB. Techniques based on the use of only one of these antibodies, specific for either the B subunit or the M subunit, were initially introduced as described in U.S. Pat. Nos. 4,067,775; 4,012,285; 3,932,221; and as described in an article by R. Roberts, et al. in The Lancet 319 (1977). These methods were found to measure other isoenzymes of CK besides CK-MB and found to produce erroneous results. Physicians who use these methods are therefore required to exercise great care when using these methods for the diagnosis of AMI.
Several new "sandwich" approaches have been proposed to overcome the problems encountered due to interference from other CK isoenzymes when antibodies specific to CK-MB or antibodies specific to only CK-MM or CK-BB are used. U.S. Pat. No. 4,353,982 describes a triple antibody technique for measuring CK-MB. In such a method, one unlabeled antibody reacts with one subunit of CK-MB, for example, the M subunit. The other antibody, that is labeled with a signal generating molecule, reacts with the second subunit of CK-MB, in thise case, the B subunit, forming a sandwich around CK-MB. CK-MB bound to the unlabeled antibody described above is precipitated from the reaction mixture by a third antibody, which is specifically formed against the IgG of the animal species used to make the unlabeled antibody. It has been shown that this technique is still prone to unacceptable interference from the other isoenzymes of CK in clinical situations, if the reaction is carried out in one step.
A two step method including precipitating the unlabeled antibodies after reaction with the specimen and diluting the undesirable impurities through the use of a wash liquid has been described in the copending commonly assigned patent application U.S. Ser. No. 165,001. The method involves a two-site sandwich technique which uses two cross-reactive antibodies in a two step procedure. This method has proved to be very accurate, and a test kit incorporating this method has been on sale in the United States for more than three years. Recently a one step method for utilizing two monoclonal antibodies raised against CK-BB and CK-MM and using immunoenzymatic labeling has been commercialized; this is the "Tandem CK-MB" test by Hybritech, Inc.
Immunometric methods using two polyclonal antibodies and two steps, such as is described in U.S. patent application Ser. No. 165,001, have eliminated many of the interference problems resulting from the other isoenzymes of CK. However, the procedures are lengthy requiring upwards to two to three hours to perform if one wants to detect small elevations of CK-MB accurately. In addition, non-specific reactions resulting from the use of iodinated tracers, and mobile particulate solid-phases, and intereference from other components present in the reaction mixture were found to occur, resulting in erroneous values. The immunoenyzomatic method using two monoclonal antibodies is also lengthy and was found to have interference with CK-BB.IgG.
It has recently become apparent that many of the patients who suffer AMI, and are at higher risk of mortality, have only a small increase in CK-MB. They are also generally more difficult to diagnose by other techniques such as electrocardiography. Many of these patients, if correctly diagnosed, could benefit from aggressive treatment involving open-heart surgery, administration of thrombolytic drugs or balloon catheterization. Thus, a need exists for accurate tests for CK-MB which could rapidly detect a small elevation of CK-MB.
The present invention is generally useful for all patients but is specifically aimed at rapidly and accurately detecting the small elevations of CK-MB indicative of AMI. In the instant invention a two-site two step immunoradiometric procedure for CK-MB is used in conjunction with a novel process and novel composition. The invention greatly increases the specific response due to CK-MB and simultaneously reduces non-specific reactions resulting from the tracer or sample matrix.